No carbon storage in growth-limited trees in a semi-arid woodland

Plant survival depends on a balance between carbon supply and demand. When carbon supply becomes limited, plants buffer demand by using stored carbohydrates (sugar and starch). During drought, NSCs (non-structural carbohydrates) may accumulate if growth stops before photosynthesis. This expectation is pervasive, yet few studies have combined simultaneous measurements of drought, photosynthesis, growth, and carbon storage to test this. Using a field experiment with mature trees in a semi-arid woodland, we show that growth and photosynthesis slow in parallel as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\psi }_{{pd}}$$\end{document}ψpd declines, preventing carbon storage in two species of conifer (J. monosperma and P. edulis). During experimental drought, growth and photosynthesis were frequently co-limited. Our results point to an alternative perspective on how plants use carbon that views growth and photosynthesis as independent processes both regulated by water availability.

Provide details about the data collection procedure, including the instruments or devices used to record the data (e.g. pen and paper, computer, eye tracker, video or audio equipment) whether anyone was present besides the participant(s) and the researcher, and whether the researcher was blind to experimental condition and/or the study hypothesis during data collection.
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The Los Alamos Survival/Mortality (SUMO) experiment22,31-33 and MDB site20,34 have been described in detail previously by others. Briefly, the SUMO and MDB sites are located near Los Alamos, New Mexico USA, at elevations of 2150m and 2140m, respectively in piñon-juniper woodland just below the Pinus ponderosa forest ecotone. Pinus edulis and Juniperus monosperma dominate both sites, although scattered individuals of Quercus gambelli, P. ponderosa, J. deppeana and J. scopulorum can also be found at the SUMO site. A volcanic tuff parent material sits below the Hackroy clay loam soils that are found at both sites and can be found at depths ranging from 40-80 cm. The growing season occurs between April and October. Average 30-year temperature and precipitation were 10.1 C and 360mm, respectively. At both sites, trees growing naturally in the field (i.e., not planted but naturally recruited) were selected for observation and experimental study. At SUMO, below-canopy precipitation removal structures and open-top heating chambers were installed during June 2012. A total of 64 individuals of P. edulis and J. monosperma (32 trees per species) growing in the ground were selected and placed into one of five treatments (5-7 trees per species in each), however due to a lack of growth data, only four treatments are considered in this paper. The ambient treatment consisted of trees exposed to ambient temperature and precipitation. The heat treatment was implemented by placing open top chambers around selected trees to create an average increase of 4.8°C above ambient temperatures. Drought trees were exposed to ambient temperatures within a precipitation removal structure that diverted~45% of precipitation away from these trees. Heat + Drought trees were exposed to both the 4.8°C temperature increase and the precipitation removal. Continuous measurement of site climatic conditions using two weather stations, in addition to within-chamber measurements, allowed control of chamber conditions using heating and air-conditioning units. In general, all measurements were made on the same trees such that comparisons of growth, photosynthesis, water potential, or NSC are robust. However, every parameter was not measured on every tree. Where all measurements in an analysis are not present for every tree in the study, those trees are excluded from that analysis. All trees assess in this study at SUMO had measurements of growth, photosynthesis, water potential, and NSC. At the MDB site, five trees each of P. edulis and J. monosperma were selected for long term monitoring in March 1992, and two additional P. edulis trees were added in 1994. In 2003, all seven measured P. edulis trees died from drought and bark beetle attack, and five surviving replacements were selected in 2004. Measurements from one tree were switched to another in 2008. Several J. monosperma were added to measurements in subsequent years: five in 2007, and three in 2015.
12 Juniperus monosperma, 13 Pinus edulis. All trees were reproductively mature and ranged in size from 0.5 m to 5.5m tall and crown widths that ranged from 1-5 m. Trees included in the study were selected based on two criteria: large enough to support several physiological measurements for multiple years, and no less than 10m from the nearest edge of drought structures.
Mesocosm experiments on mature trees are rare because of difficulties in exposing such large organisms to experimental conditions. Our experiment simulated drought and heat conditions in the field, which is inherently limited by species density (e.g., we could not move trees into our plots). In this case, sample size was not a limiting factor (n>10) for the statistical tests we used (primarily linear regression, F-tests, and Analysis of Variance, given the number of factor levels in our models (2).

Radial Growth Measurements and Calculations
At SUMO, tree radial growth was measured as outlined by Manrique-Alba et al. 33. Briefly, from May through September in 2013 and 2014, a linear variable displacement transducer (LVDT) was attached to the upper bole of 11 individuals of J. monosperma and 12 individuals of P. edulis using a rectangular frame that was attached directly to the tree using screws. Trees were selected from Heat, Heat + Drought, Drought, and Ambient treatments. Dead bark was gently removed from the site where the sensor contacted the tree, though a thin layer was left as to protect the phloem and prevent water loss.
Data from the LVDT is a relative metric of change in diameter over time. Upon installation, each LVDT was set to "zero" the night of the first day of the measurement period before any growth occurred. This established the reference point from which measured growth would deviate. Because diurnal fluxes of stem diameter make calculating growth difficult, we considered growth initiation to have occurred when the maximum stem diameter exceeded that of the previous day's maximum, for each tree. When maximum stem diameter did not exceed the previous day's maximum, we considered growth to have stopped. No direct measurements of growth were made on trees at the MDB site. Water potential and Photosynthesis At SUMO, xylem water potential and foliar gas exchange were determined for 11 individuals of J. monosperma and 12 individuals of P. edulis (2011 -2017). Two twig samples were collected every three months from each tree before sunrise and the xylem water tension was measured using a Scholander pressure chamber (PMS Instruments, Albany, OR, USA). The level of water stress for each tree was quantified as the average predawn water potential (!_pd) of both stems. At MDB, xylem water potential was measured nature portfolio | reporting summary

March 2021
Timing and spatial scale Data exclusions Reproducibility Randomization Blinding Did the study involve field work?

Yes No
Field work, collection and transport Field conditions every month between 1992 -2016. Measurements were made on 5-6 individuals of J. monosperma from 1992 -2012, and 11-14 individuals of J. monosperma from 2013 -2016. Between 1992 -2016, water potential measurements were made on 5-7 individuals of P. edulis. At SUMO only, net photosynthesis and gas exchange were measured on the south-facing, sun-exposed side of each tree using a Li-Cor LI-6400 (Lincoln, NE, USA). Needles from each tree were measured under chamber conditions set of 380ppm CO2, 1500 mol m( -2) s^(-1) light-saturating photosynthetic photon flux density (PPFD), temperatures between 20-25°C, and 0% relative humidity. These conditions closely matched those of the outside environment, where temperatures ranged from 13 -30°C and 750 -1800 mmol m^(-2) s^(-1) PPFD. After two minutes of steady-state gas exchange, measurements were recorded, and needle samples were collected to determine leaf area. Gas exchange data was corrected using leaf area measurements made on a Li-Cor LI3100C area meter. No gas exchange measurements were made on the trees at MDB.

Non-Structural Carbohydrates
Beginning on March 14th, 2012, and ending on October 13th, 2016 at SUMO, leaf and stem (twig; phloem and bark) tissue samples were collected for each tree four times each year to capture seasonal changes associated with spring dormancy break, midsummer drought, monsoon wet-season, and post-monsoon dry-down. Stem samples were from recent growth, dated from 0 to 5 years old for P. edulis. Bole and root samples (also including bark) were collected using an increment borer once per year during the dry season, in June. All samples were collected between 11:30 and 13:00, mitigating any influence of diurnal variation in NSC on our measurements (Gersony et al. 2020, Tixier et al. 2018. Upon collection, samples were placed into liquid nitrogen and transported to the lab in dry ice. Samples were kept stored at -70°C until analysis, when they were microwaved for 5 min at 800W and placed in a drying oven for 48h at 65°C. All samples were ground using a ball mill and woody tissues were preground using a Wiley Mini-Mill. To assay NSC, we used the protocol outlined by Dickman et al. 35, as developed from the methods of Hoch et al. 36. This method has been verified to produce reasonably accurate and precise measurements of NSC, defined as glucose, fructose, sucrose, and starch.37 12mg of finely ground sample was placed into a deep-well plate with 1.6 mL deionized water and placed into a 100C water bath for 1h. An NAD-linked enzymatic assay was used in combination with spectral assessment at 340 nm for NSC quantification. To analyze NSCs at the whole-tree or canopy scales we averaged NSC concentrations from each respective tissue. For example, to calculate canopy NSC, sugar, and starch, we averaged the NSC from stem and needle tissues. A similar approach was used to estimate wholetree NSC for June. No NSCs were measured on the trees at MDB.
All treatments were initiated on 11 June 2012. Growth was measured daily, beginnin May 1st and ending Sept 30th 2013, and from May 1st to Sept 8th 2014. NSC was measured monthly every year from March 14 2012 to Oct 13 2016. Water potential was measured monthly from March 10 2011 -May 9 2017. Photosynthesis was measured monthly from April 4 2012 -Mar 21 2017. This relatively frequent sampling for the duration of the experiment allowed us to capture any seasonal effect on these parameters as well as fully characterize the physiological response of these trees to experimental treatments. All samples were collected at the scale of individual trees.
Data was excluded from this manuscript only if it was determined to be an outlier in the analysis. Outlier criteria was set using 3x Cook's distance, as described in our manuscript. The rational for this was to remove any overwhelming effect on regression coefficients on the main results. this is a standard statistical procedure to remove data points that appear to be erroneous.
Due to the high cost of the experiment and relatively long duration, no attempts to reproduce this experiment have occurred. However, many similar drought experiments have since occurred both in this ecosystem (i.e., Sevilleta National Wildlife Refuge) and in the tropics.
Plots were not randomly placed since construction constraints dictated where specific treatments could be installed. Within plots, ttrees that fit the size criteria outlined above were randomly selected and placed into their respective treatments.
Blinding was not relevant to this study since it is inherently observational.
The Los Alamos Survival/Mortality (SUMO) experiment22,31-33 and MDB site20,34 have been described in detail previously by others. Briefly, the SUMO and MDB sites are located near Los Alamos, New Mexico USA, at elevations of 2150m and 2140m, respectively in piñon-juniper woodland just below the Pinus ponderosa forest ecotone. Pinus edulis and Juniperus monosperma dominate both sites, although scattered individuals of Quercus gambelli, P. ponderosa, J. deppeana and J. scopulorum can also be found at the SUMO site. A volcanic tuff parent material sits below the Hackroy clay loam soils that are found at both sites and can be found at depths ranging from 40-80 cm. The growing season occurs between April and October. Average 30-year temperature and precipitation were 10.1 C and 360mm, respectively. At both sites, trees growing naturally in the field (i.e., not planted but naturally recruited) were selected for observation and experimental study. At SUMO, below-canopy precipitation removal structures and open-top heating chambers were installed during June 2012. A total of 64 individuals of P. edulis and J. monosperma (32 trees per species) growing in the ground were selected and placed into one of five treatments (5-7 trees per species in each), however due to a lack of growth data, only four treatments are considered in this paper. The ambient treatment consisted of trees exposed to ambient temperature and precipitation. The heat treatment was implemented by placing open top chambers around selected trees to create an average increase of 4.8°C above ambient temperatures. Drought trees were exposed to ambient temperatures within a precipitation removal structure that diverted~45% of precipitation away from these trees. Heat + Drought trees were exposed to both the 4.8°C temperature increase and the precipitation removal. Continuous measurement of site climatic conditions using two weather stations, in addition to within-chamber measurements, allowed control of chamber conditions using heating and air-conditioning units. In general, all measurements were made on the same trees such that nature portfolio | reporting summary

March 2021
Location Access & import/export Disturbance Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Dating methods comparisons of growth, photosynthesis, water potential, or NSC are robust. However, every parameter was not measured on every tree. Where all measurements in an analysis are not present for every tree in the study, those trees are excluded from that analysis. All trees assess in this study at SUMO had measurements of growth, photosynthesis, water potential, and NSC. At the MDB site, five trees each of P. edulis and J. monosperma were selected for long term monitoring in March 1992, and two additional P. edulis trees were added in 1994. In 2003, all seven measured P. edulis trees died from drought and bark beetle attack, and five surviving replacements were selected in 2004. Measurements from one tree were switched to another in 2008. Several J. monosperma were added to measurements in subsequent years: five in 2007, and three in 2015.

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Los Alamos, New Mexico, USA (elev. 2140 All samples were collected with explicit permission from Los Alamos National Laboratory and the US Department of Energy. Site access at SUMO is highly restricted, and permits allowing the experiment and access to the field site were awarded via DOE contract #: AC52-06NA25396. This research significantly disturbed the ecosystem in which it was performed. This disturbance was necessary, as the primary aim of this experiment was to understand the response of this ecosystem to experimental disturbance (here, heat and drought).
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